The International Agency for Research on Cancer evaluates acetaldehyde as "possibly carcinogenic to humans" while the U.S. Dept. of Health and Human Services states that acetaldehyde is "reasonably anticipated to be a human carcinogen." These evaluations are based on convincing carcinogenicity studies that clearly demonstrate that acetaldehyde causes respiratory tract tumors in rats and hamsters when administered by inhalation. There is great potential for extensive human exposure to acetaldehyde. Cigarette smoke contains about 1 mg of acetaldehyde per cigarette, which is far greater than the concentrations of other carcinogens such as benzo[a]pyrene or tobacco-specific nitrosamines, widely assumed to be important as causes of cancer in smokers. Acetaldehyde is the major metabolite of ethanol and can reach significant concentrations in people who drink alcoholic beverages, especially if they are deficient in certain forms of aldehyde dehydrogenase. Acetaldehyde has been implicated as a cause of several cancer types associated with alcohol consumption. Human exposure to acetaldehyde can also occur in occupational and environmental settings. In spite of extensive human exposure to this carcinogen, little is known about its DNA adducts, which are likely central to its carcinogenic activity. We have recently characterized several DNA adducts of acetaldehyde including the Schiff base N2-ethylidenedeoxyguanosine, two diastereomeric l,N2-propanodeoxyguanosine adducts, three diastereomeric N2-dimethyldioxanyldeoxyguanosine adducts, and an interstrand G-G crosslink. These data promise to open new avenues of understanding of acetaldehyde carcinogenesis. Therefore, we propose to extend these studies in the present proposal. Our specific aims are: 1) Develop sensitive mass spectrometric methods to detect acetaldehyde-DNA adducts. These methods will be applied for analysis of acetaldehyde-DNA adducts in vitro, in laboratory animals exposed to acetaldehyde, and in humans (Specific Aims 2, 4, and 5); 2) Investigate the formation and stereochemistry of acetaldehyde-DNA adducts in vitro, using concentrations of acetaldehyde that are likely to be achieved in vivo; 3) Synthesize oligonucleotides containing the acetaldehyde-DNA crosslink adduct and investigate its repair and mutagenicity; 4) Quantify the formation and persistence of acetaldehyde-DNA adducts in rats or mice exposed to acetaldehyde or ethanol; 5) Quantify acetaldehyde-DNA adducts in white blood cells of humans exposed to acetaldehyde. These studies will be carried out in smokers, non-smokers, casual drinkers, and people with alcohol dependence. The results of the research proposed here will provide critical data, which will be the framework for testing our hypothesis that acetaldehyde is a human carcinogen.